CN106421076B - A kind of preparation method and application of bamboo willow bark anti-inflammatory activity extract - Google Patents
A kind of preparation method and application of bamboo willow bark anti-inflammatory activity extract Download PDFInfo
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Abstract
The present invention provides a kind of preparation method and application of bamboo willow bark anti-inflammatory activity extract, using bamboo willow bark as raw material, it using ethyl alcohol as solvent, is extracted using ultrasonic technology, extracting solution is concentrated under reduced pressure into no ethyl alcohol residual, appropriate distilled water dissolution is added, ethyl acetate extraction, extract liquor are concentrated under reduced pressure into certain volume, are dried in vacuo, it is then purified using decompression silica gel column chromatography and Sephadex LH-20 gel column, obtains bamboo willow ethyl acetate extract.Experiment shows that bamboo willow ethyl acetate extract paraxylene causes mouse ear swelling, swollen hyperplasia of rat granuloma chronic inflammation and mouse acetic acid twisting to have preferable inhibitory activity, it can be compounded directly as single preparations of ephedrine or with other components is used for acute and chronic inflammatory disease, the especially treatment of the psoriasis as caused by chronic inflammation.Present invention success extracts anti-inflammatory active ingredient from bamboo willow, is that application of the bamboo willow in medical product field lays the foundation.
Description
Technical field
The present invention relates to a kind of preparation method and application of bamboo willow bark anti-inflammatory activity extract, which is isolated from bamboo willow
Skin ethyl acetate extract has anti-inflammatory and antalgic activity, belongs to the extracting and developing purification art of active ingredient of natural product.
Background technique
Bamboo willow (Salix ssp.) is also known as U.S. bamboo willow, is Salicaceae (Saliaceae) Salix (Salix) plant, is beauty
State trembles with fear the excellent strain (prince is at 2008) of willow, korean willow and osier willow hybridization, in numerous excellent of inheriting tradition sallow
The advantages that growth is fast, material is good, resistance (strong etc., 2009) is shown while moral character, is common fast-growing timber forests tree
Kind and Tree Species as Bio-energy (Xu Keshun, 2011).
In recent years, bamboo willow cultivated area expands rapidly in China, and according to statistics, woods in blocks is more than 1300hm2(Hu Guotao
Deng 2016), the storage level of bamboo willow is abundant, but the industrialization of current bamboo willow is still immature, rarely has deep processed product and high added value
Product.And willow is belonged to as medicinal with a long history, just record that " for Liu Weiben through low-grades, property is bitter in Ming Dynasty's Compendium of Material Medica
It is cold, nontoxic, geomantic omen yellow subcutaneous ulcer, sore, carbuncle and painful swelling, phlegm heat gonorrhea disease, the diseases such as arthritis with fixed pain caused by dampness disease can be treated ", and its each section can all be used as medicine, and be
" whole body is all precious ".Modern chemistry and pharmacological activity in relation to sallow studies have shown that its chemical component include flavonoids,
Phenolic glycoside class, Phenylpropanoid Glycosides class, quinones, steroid, terpene, organic acid etc., have anti-inflammatory, analgesic, anti-oxidant, antibacterial, anti-mutagenesis,
The pharmacological actions such as decompression, diuresis (Liu Moxiang etc., 1997；Envelope Silan etc., 2001；Zhao Hongjuan etc., 2010；Moso bamboo monarch etc., 2013).
The bamboo willow to belong to is pretended, although being rarely reported at present to research relevant in terms of its chemical component and bioactivity.But it is existing
Research shows that bamboo willow shows bioactivity similar with congener, there are huge potentiality to can be developed into the medicine of high added value
Product.
CN105237594A discloses a kind of preparation method that salicin is extracted from bamboo willow bark, and this method is: by bamboo willow
After bark crushes, using continuous microwave drying method, bamboo willow bark powder is made, must be extracted after being extracted with deionized water ultrasound adverse current
Liquid, extracting solution is purified, is concentrated, obtains salicin after drying.Only the salicin in bamboo willow is extracted in the invention, and
Research is not extracted to other effective components in bamboo willow.And do not have in the prior art by willow or bamboo willow ethyl acetate extract yet
Relevant report for curing psoriasis.
Summary of the invention
The purpose of the present invention is the deficiency for correlative studys such as existing bamboo willow chemical component, bioactivity and with bamboo
Willow is the blank of the medical product of raw material exploitation, provides a kind of method that anti-inflammatory and antalgic activity component is extracted from bamboo willow bark, that is,
The preparation method of bamboo willow bark anti-inflammatory activity extract, and have studied application of the active component in medical product field.
In order to achieve the above objectives,
The first aspect of the invention is to provide a kind of preparation method of bamboo willow bark anti-inflammatory activity extract, feature
It is that, by bamboo willow bark powder, 5 to 20 times of EtOH Sonicates of use extract 1~5 time, and each 30-120min obtains extracting solution；Extracting solution is subtracted
Pressure is concentrated into no ethyl alcohol residual, and distilled water dissolution is added, is extracted with ethyl acetate, through both obtaining after purification twice.
Specific step is as follows:
A) extract: ethanol solution is added according to solid-liquid ratio 1:5~1:20 in bamboo willow bark powder, 10~40 mesh of granularity, and ultrasound mentions
Taking condition is 9~18KHz of frequency, and 20~50 DEG C of temperature, 30~120min of time, ultrasonic number 1~5 time obtains extracting solution；
Extracting solution is concentrated under reduced pressure into no ethyl alcohol residual, and appropriate distilled water dissolution is added, is extracted 1~3 time with 1~3 times of amount ethyl acetate, extraction
It takes liquid to be concentrated under reduced pressure, is dried in vacuo (600~1000Pa, 40~50 DEG C, 120~180min)；Obtain crude extract powder；
B) VLC (decompression silica gel column chromatography) purifying: 100~300 mesh silica gel wet method dress posts, diameter height ratio 1:5, step A) gained
Powder dry method loading removes the low polar impurity in acetic acid ethyl ester extract using petroleum ether as eluant, eluent；With petroleum ether and acetic acid
The mixed liquor that ethyl ester volume ratio is 2:8 is eluant, eluent, and eluent is concentrated under reduced pressure into certain volume, be dried in vacuo (600~
1000Pa, 40~50 DEG C, 120~180min), obtain the bamboo willow bark anti-inflammatory activity extract powder of initial purification；
C) Sephadex LH-20 purify: gel column diameter height ratio 1:18, step B) gained powder with a small amount of chloroform-methanol mix
Liquid dissolution is closed, wet process loading, with chloroform-methanol (1:1) for eluant, eluent, flow velocity is 0.3~0.5BVh-1, isocratic elution, HPLC
Combining data detection, the bamboo willow bark activity extract solution with anti-inflammatory and antalgic activity for the pigment that is removed；
D) dry and save: solution step C) is concentrated under reduced pressure into certain volume, be dried in vacuo (600~1000Pa, 40~
50 DEG C, 120~180min), after sterilized, sterilization, obtain bamboo willow bark anti-inflammatory activity extract powder.
Preferably, the step A) in ethanol solution volume fraction be 70%, raw material granularity be 24 mesh, solid-liquid ratio 1:
10, supersonic frequency 18KHz, 30 DEG C of ultrasonic temperature, ultrasonic time 60min, ultrasonic number 3 times.The use of extractant (ethyl acetate)
Amount is identical as the dosage of distilled water used in dissolution extract, extracts 3 times, is dried in vacuo 150min under the conditions of 800Pa, 45 DEG C；
Preferably, the step B) in silica gel granularity be 200~300 mesh；Using petroleum ether as eluent 2BV, remove
Low polar impurity；Using volume ratio for 2:8 petroleum ether and ethyl acetate mixtures as eluent 3BV, merge eluent, in
It is concentrated under reduced pressure under the conditions of 40 DEG C, is dried in vacuo 120min under the conditions of 800Pa, 45 DEG C；
Preferably, the step C) wet process loading sample concentration be 50mg/mL, flow velocity 0.4BVh-1, 3BV is eluted,
Eluent after collecting 2h；The chromatographic condition of HPLC detection is 0-20min, 20-45% methanol-water, 20-70min, 45-80% first
Alcohol water, 70-80min, 80-100% methanol-water, 80-90min, 100% methanol, flow velocity 1ml/min, Detection wavelength are
220nm, column temperature: 25 DEG C, YMC-pack ODS A chromatographic column (250 × 4.6mm, 5 μm)；
Preferably, the step D) vacuum drying condition be 1000Pa, 40 DEG C, 120min；The disinfection, sterilizing include
One or more of radiation sterilization, vapor sterilizing, hot-air sterilizing, 80% ethanol solution combination.
The research of the invention finds that bamboo willow bark anti-inflammatory activity extract paraxylene causes mouse ear swelling, swollen hyperplasia of rat granuloma
Chronic inflammation and mouse acetic acid twisting have preferable inhibitory activity.Show that the extract can directly be made by human trial
It is single preparations of ephedrine or compounds the treatment for acute and chronic inflammatory disease with other components, especially as caused by chronic inflammation
The treatment of psoriasis.
Therefore, the second aspect of the present invention provides the preparation containing bamboo willow bark anti-inflammatory activity extract, anti-inflammatory by bamboo willow bark
Activity extract and pharmaceutically acceptable carrier or diluent composition.The preparation be tablet, dragee, capsule, injection,
Solution, lotion, ointment, emulsifiable paste, aerosol or suppository.
Third aspect of the present invention provides bamboo willow bark anti-inflammatory activity extract or contains bamboo willow bark anti-inflammatory activity extract
Preparation, the application in medical product field.Preferably, the application in the drug of preparation prevention and treatment psoriasis.
The pharmaceutically acceptable carrier or diluent refer to the pharmaceutical carrier of pharmaceutical field routine, selected from filler,
One or more of adhesive, disintegrating agent, lubricant, suspending agent, wetting agent, solvent, surfactant or corrigent.It is described
Filler is selected from starch, sucrose, lactose, mannitol, sorbierite, xylitol, microcrystalline cellulose or glucose etc.；Described adhesive
Selected from cellulose derivative, alginates, starch, dextrin, gelatin or polyvinylpyrrolidone etc.；It is fine that the disintegrating agent is selected from crystallite
Tie up element, sodium carboxymethyl starch, crosslinked polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose or croscarmellose sodium；
The lubricant is selected from stearic acid, polyethylene glycol, calcium carbonate, sodium bicarbonate, superfine silica gel powder, talcum powder or magnesium stearate；It is described
Suspending agent is selected from superfine silica gel powder, beeswax, cellulose, solid polyethylene glycol；The wetting agent is selected from glycerol, Tween 80, ethyoxyl
Rilanit special or lecithin；The solvent be selected from ethyl alcohol, liquid polyethylene glycol, isopropanol, Tween-80, glycerol, propylene glycol or
Vegetable oil, the vegetable oil are selected from soybean oil, castor oil, peanut oil, ready-mixed oil etc.；The surfactant is selected from dodecyl
Benzene sulfonic acid sodium salt, stearic acid, one poiyoxypropylene copolymer of polyoxyethylene, fatty acid sorbitan or polysorbate (tween) etc.；It is described
Corrigent is selected from Aspartame, Sucralose, essence, citric acid or saccharin sodium.
The invention has the following beneficial effects:
1. the invention using fast-growing timber forests tree species and the bamboo willow of Tree Species as Bio-energy as research object, for the first time
Ethyl acetate extracting method is carried out to bamboo willow bark and carries out exploratory development, bamboo willow bark is obtained and carries out ethyl acetate extract, i.e. this hair
Bright bamboo willow bark anti-inflammatory activity extract.The application for being bamboo willow in medical product field lays the foundation, and facilitates bamboo willow added value
Raising.
2. the present invention has carried out pharmacology and toxicity research to bamboo willow bark ethyl acetate extract, it was demonstrated that the extract can be straight
The treatment for acute and chronic inflammatory disease is connect as single preparations of ephedrine or compounded with other components, is especially led by chronic inflammation
The treatment of the psoriasis of cause.
3. bamboo willow bark ethyl acetate extract anti-inflammatory and antalgic activity of the present invention is high, have no toxic side effect, safety is good, and uses
Whole low-temperature operation technique guarantees extract chemical stability, and solvent for use can be recycled, and is suitble to industrialized production.
Detailed description of the invention
Fig. 1 is the step A gained sample HPLC chromatogram in bamboo willow bark anti-inflammatory activity method for preparing extractive of the present invention.
Fig. 2 is the step B gained sample HPLC chromatogram in bamboo willow bark anti-inflammatory activity method for preparing extractive of the present invention.
Fig. 3 is the step C gained ethyl acetate extract in bamboo willow bark anti-inflammatory activity method for preparing extractive of the present invention
HPLC chromatogram.
Fig. 4 is that the bamboo willow bark anti-inflammatory activity extract of embodiment 1, comparative example 1 and comparative example 2 is swollen to mouse ear
Swollen, swollen hyperplasia of rat granuloma and mouse writhing response inhabitation rate result.
Specific embodiment
Technical solution of the present invention and its generated technical effect are carried out below with reference to specific test method and attached drawing
Further elucidated above, the following description is only intended to explain the invention, but the present invention is limited in any way, based on this
It is any made by invention introduction to transform or replace, it all belongs to the scope of protection of the present invention.
Embodiment 1
A) extract: precision weighs the bamboo willow bark powder 100g for smashing it through 24 meshes, and 70% ethyl alcohol that ten times of amounts are added is molten
Liquid is as Extraction solvent, and ultrasonic wave extraction 3 times, each 60min, ultrasonic temperature is 30 DEG C, and filtering, extracting solution merges, and 45 DEG C subtract
Pressure is concentrated into no ethyl alcohol residual, the sample solution that 100mg/mL is made in the dissolution of 80mL distilled water is added, with the acetic acid of same volume
Ethyl ester extracts 3 times, and extract liquor merges, and 40 DEG C are concentrated under reduced pressure into certain volume, are dried in vacuo under the conditions of 800Pa, 45 DEG C
150min；Sample chromatogram figure is as shown in Figure 1.
B 200-300 mesh silica gel wet method dress post, diameter height ratio 1:5, by step A) VLC (decompression silica gel column chromatography) purifying: are used
Gained powder dry method loading removes low polar impurity using petroleum ether as eluent 2BV；It is the petroleum ether of 2:8 with volume ratio
It is eluent 3BV with ethyl acetate mixtures, merges eluent, be concentrated under reduced pressure under the conditions of 40 DEG C, in 800Pa, 45 DEG C
Under the conditions of be dried in vacuo 120min；Sample chromatogram figure is as shown in Figure 2.
C) Sephadex LH-20 is purified: gel column diameter height compares for 1:18, is 1 by powder stone volume ratio obtained by step B:
50mg/mL sample solution is made in 1 chloroform and methanol solution dissolution, is for the chloroform of 1:1 and the mixed liquor of methanol with volume ratio
Eluant, eluent, by 0.4BVh-1Flow velocity elute 3BV, collect 2h after eluent, HPLC detection (chromatographic condition 0-20min,
20-45% methanol-water, 20-70min, 45-80% methanol-water, 70-80min, 80-100% methanol-water, 80-90min, 100% first
Alcohol, flow velocity 1ml/min, Detection wavelength 220nm, column temperature: 25 DEG C, YMC-pack ODS A chromatographic column (250 × 4.6mm, 5 μ
M)), be removed the bamboo willow ethyl acetate extract solution of pigment；Chromatogram is as shown in Figure 3.
D) dry and preservation: solution is concentrated under reduced pressure into certain volume, and 1000Pa, is dried in vacuo 120min by 40 DEG C, and radiation is gone out
Bacterium finally saves bamboo willow ethyl acetate extract in powder form.
Embodiment 2
A) extract: precision weighs the bamboo willow bark powder 100g for smashing it through 40 meshes, and 70% ethanol solution that 5 times of amounts are added is made
For solvent, ultrasonic wave extraction 4 times, each 45min, ultrasonic temperature is 40 DEG C, and filtering, extracting solution merges, and 45 DEG C are concentrated under reduced pressure into
The sample solution that 100mg/mL is made in the dissolution of 80mL distilled water is added in no ethyl alcohol residual, and the ethyl acetate measured with 2 times extracts 3 times,
Extract liquor merges, and 40 DEG C are concentrated under reduced pressure into certain volume, are dried in vacuo 120min under the conditions of 1000Pa, 40 DEG C；
B 200-300 mesh silica gel wet method dress post, diameter height ratio 1:5, by step A) VLC (decompression silica gel column chromatography) purifying: are used
Gained powder dry method loading removes low polar impurity using petroleum ether as eluent 2BV；It is the petroleum ether of 2:8 with volume ratio
It is eluent 3BV with ethyl acetate mixtures, merges eluent, be concentrated under reduced pressure under the conditions of 40 DEG C, in 1000Pa, 40 DEG C
Under the conditions of be dried in vacuo 120min；
C) Sephadex LH-20 is purified: gel column diameter height compares for 1:18, is 1 by powder stone volume ratio obtained by step B:
50mg/mL sample solution is made in 1 chloroform and methanol solution dissolution, is for the chloroform of 1:1 and the mixed liquor of methanol with volume ratio
Eluant, eluent, by 0.5BVh-1Flow velocity elute 3BV, collect 1.6h after eluent, HPLC detect (chromatographic condition 0-
20min, 20-45% methanol-water, 20-70min, 45-80% methanol-water, 70-80min, 80-100% methanol-water, 80-90min,
100% methanol, flow velocity 1ml/min, Detection wavelength 220nm, column temperature: 25 DEG C, YMC-pack ODS A chromatographic column (250 ×
4.6mm, 5 μm)), the bamboo willow ethyl acetate extract solution for the pigment that is removed；
D) dry and preservation: solution is concentrated under reduced pressure into certain volume, and 1000Pa, is dried in vacuo 120min by 40 DEG C, and radiation is gone out
Bacterium finally saves bamboo willow ethyl acetate extract in powder form.
Embodiment 3
A) extract: precision weighs the bamboo willow bark powder 100g for smashing it through 15 meshes, and 70% ethanol solution of 20 times of amounts is added
As solvent, ultrasonic wave extraction 3 times, each 45min, ultrasonic temperature is 50 DEG C, and filtering, extracting solution merges, 45 DEG C of reduced pressures
It is remained to no ethyl alcohol, the sample solution that 100mg/mL is made in the dissolution of 80mL distilled water is added, is extracted with the ethyl acetate of same volume
It takes 3 times, extract liquor merges, and 40 DEG C are concentrated under reduced pressure into certain volume, are dried in vacuo 180min under the conditions of 600Pa, 40 DEG C；
B 200-300 mesh silica gel wet method dress post, diameter height ratio 1:5, by step A) VLC (decompression silica gel column chromatography) purifying: are used
Gained powder dry method loading removes low polar impurity using petroleum ether as eluent 2BV；It is the petroleum ether of 2:8 with volume ratio
It is eluent 3BV with ethyl acetate mixtures, merges eluent, be concentrated under reduced pressure under the conditions of 40 DEG C, in 600Pa, 40 DEG C
Under the conditions of be dried in vacuo 150min；
C) Sephadex LH-20 is purified: gel column diameter height compares for 1:18, is 1 by powder stone volume ratio obtained by step B:
50mg/mL sample solution is made in 1 chloroform and methanol solution dissolution, is for the chloroform of 1:1 and the mixed liquor of methanol with volume ratio
Eluant, eluent, by 0.5BVh-1Flow velocity elute 3BV, collect 1.6h after eluent, HPLC detect (chromatographic condition 0-
20min, 20-45% methanol-water, 20-70min, 45-80% methanol-water, 70-80min, 80-100% methanol-water, 80-90min,
100% methanol, flow velocity 1ml/min, Detection wavelength 220nm, column temperature: 25 DEG C, YMC-pack ODS A chromatographic column (250 ×
4.6mm, 5 μm)), the bamboo willow ethyl acetate extract solution for the pigment that is removed；
D) dry and preservation: solution is concentrated under reduced pressure into certain volume, and 1000Pa, is dried in vacuo 120min by 40 DEG C, and radiation is gone out
Bacterium finally saves bamboo willow ethyl acetate extract in powder form.
Comparative example 1
By the A in embodiment 1) and D) corresponding powder is made in step.
Comparative example 2
By the A in embodiment 1), B) and D) corresponding powder is made in step.
Experimental example 1
Bamboo willow bark anti-inflammatory activity extract of the present invention inhibits xylene-induced ear swelling in mice test:
18~22g of weight male mice in kunming 50 is taken, is randomly divided into 5 groups, every group 10, including example 1 group (with
10%DMSO dissolution, it is 10mg/mL test sample solution that concentration, which is made), 1 group of comparative example (is dissolved with 10%DMSO, is made
Concentration is 10mg/mL test sample solution), 2 groups of comparative example (is dissolved, it is 10mg/mL for examination that concentration, which is made, with 10%DMSO
Sample solution), positive controls (aspirin is dissolved with 10%DMSO, and it is 10mg/mL test sample solution that concentration, which is made),
Blank group (10%DMSO).Each group presses 20mLkg-1Dosage gastric infusion, one time a day, continuous 7d, last dose 30min
Afterwards, 20 μ L caused by dimethylbenzene xylene inflammation, Zuo Erwei blank are applied in mouse right ear.After 2h by mouse break cervical vertebra put to death, cut two ears, use diameter
9mm punch cuts the weighing of ears piece, subtracts the difference of left ear weight as the evaluation mark of inflammatory swelling degree using auris dextra weight
Standard, and each group mouse ear swelling inhibiting rate according to the following formula.
The experimental result of the influence of each group paraxylene induced mice ear swelling is shown in Table 1 and Fig. 4.According to the experimental results,
Compared with blank control group, each administration group shows stronger inhibition mouse ear swelling activity, and significant difference, table is presented
Bright bamboo willow ethyl acetate extract active component can significantly inhibit dimethylbenzene induced mice ear swelling；The inhibiting rate and sun of embodiment 1
Property control group is compared, unknown significance difference, but significantly increases (P < compared with the inhibiting rate of comparative example 1 and comparative example 2
0.01), and the inhibiting rate of comparative example 1 is compared with comparative example 2, and significant difference (P < 0.01) also is presented, shows bamboo
Willow acetic acid ethyl ester extract is remarkably improved the inhibitory activity of its paraxylene induced mice ear swelling after above-mentioned purification step,
Also indicate that the science and reasonability of bamboo willow bark anti-inflammatory activity method for preparing extractive of the present invention.
1 each group paraxylene induced mice ear swelling of table influence (n,)
Note: * represents the P < 0.01 compared with blank control group；Represent the P < 0.01 compared with positive controls；It represents
Compared with Example 1, P < 0.01；Δ represents the P < 0.01 compared with comparative example 1.
Experimental example 2
Bamboo willow bark anti-inflammatory activity extract of the present invention inhibits the test of swollen hyperplasia of rat granuloma chronic inflammation:
Taking weight 180 ± 10g SD rat 50, half male and half female is randomly divided into 5 groups, and every group 10, including example 1 group
(being dissolved with 10%DMSO, it is 10mg/mL test sample solution that concentration, which is made), 1 group of comparative example (it is dissolved with 10%DMSO,
It is 10mg/mL test sample solution that concentration, which is made), 2 groups of comparative example (is dissolved, it is 10mg/mL that concentration, which is made, with 10%DMSO
Test sample solution), (dexamethasone acetate is dissolved positive controls with 10%DMSO, and it is 1mg/mL test sample that concentration, which is made,
Solution) and model group (10%DMSO).
Model group rats are anesthetized with ether, in one osculum of abdominal incision under aseptic condition, with tweezers by the sterilizing of 20mg
Cotton balls is implanted under the groin of rat two sides from small notch, is sutured and is carried out disinfection with iodine solution to suture immediately.Remaining sample
Group modeling in an identical manner.Each administration group presses 20mLkg in operation next day-1Dosage gastric infusion, one time a day, continuously
After 10d, last dose 60min, the cervical vertebra that breaks puts to death rat, goes out to cut skin in former notch, by cotton balls together with surrounding connective tissue
It takes out simultaneously, rejects peripheral adipose, for 24 hours in 50 DEG C of baking oven drying, weighing.Evaluation mark using granuloma weight as degree of inflammation
Standard, and each group granuloma induced by implantation of cotton pellets inhibiting rate according to the following formula.
Each group swollen hyperplasia of rat granuloma the results are shown in Table 2 and Fig. 4.According to the experimental results, it compared with blank control group, respectively gives
Medicine group shows the stronger activity for inhibiting swollen hyperplasia of rat granuloma, and significant difference is presented, and shows bamboo willow ethyl acetate
The active component at position can inhibit the activity of swollen hyperplasia of rat granuloma；The inhibiting rate of embodiment 1 has no compared with positive controls
Significant difference, but compared with comparative example 1 and comparative example 2, the swollen hyperplasia of rat granuloma weight of example 1 group is obvious
It reduces, and significant difference (P < 0.01) is presented, show that bamboo willow bark anti-inflammatory activity extract of the present invention can significantly inhibit rat cotton
Chronic inflammation caused by ball granuloma also indicates that the science of bamboo willow bark anti-inflammatory activity method for preparing extractive of the present invention
And reasonability.
2 each group swollen hyperplasia of rat granuloma weight of table comparison (n,)
Note: * represents the P < 0.01 compared with blank control group；It represents compared with Example 1, P < 0.01.
Experimental example 3
Bamboo willow bark anti-inflammatory activity extract of the present invention inhibits the test of mouse acetic acid twisting:
Taking 18~22g of weight Kunming mouse 50, half male and half female is randomly divided into 5 groups, and every group 10, including embodiment 1
Group (is dissolved, it is 10mg/mL test sample solution that concentration, which is made) with 10%DMSO, and 1 group of comparative example (molten with 10%DMSO
Solution, it is 10mg/mL test sample solution that concentration, which is made), 2 groups of comparative example (is dissolved, concentration, which is made, is with 10%DMSO
10mg/mL test sample solution), (aspirin is dissolved positive controls with 10%DMSO, and it is 10mg/mL for examination that concentration, which is made,
Sample solution), blank group (10%DMSO).Each group presses 20mLkg-1Dosage gastric infusion, one time a day, continuous 7d, last
After 30min is administered, by 0.2mL10g-1Dosage inject 0.6% acetum (now preparing before use) to every mouse peritoneal,
It observes and records each group mouse in injection acetum 15min and writhing response (abdomen contraction indent, stretching, extension hind leg, stern occurs
Raise in portion) number, and according to the following equation calculate writhing response inhibiting rate.
Each group is shown in Table 3 and Fig. 4 to the experimental result for the influence that mouse acetic acid twisting reacts.According to the experimental results, with sky
White control group is compared, and each administration group shows the stronger activity for inhibiting mouse writhing, and significant difference is presented, and shows bamboo
Willow ethyl acetate extract active component can significantly inhibit mice pain caused by acetic acid；The inhibiting rate and positive control of embodiment 1
Group is compared, unknown significance difference, but significantly increases (P < 0.01) compared with the inhibiting rate of comparative example 1 and comparative example 2,
And the inhibiting rate of comparative example 1 is also presented significant difference (P < 0.01), shows bamboo willow acetic acid compared with comparative example 2
Ethyl ester extract is remarkably improved analgesic activities after above-mentioned purification step, also indicates that bamboo willow bark anti-inflammatory activity of the present invention
The science and reasonability of method for preparing extractive.
Influence that 3 each group of table reacts mouse acetic acid twisting (n,)
Note: * represents the P < 0.01 compared with blank control group；Represent the P < 0.01 compared with positive controls；It represents
Compared with Example 1, P < 0.01；Δ represents the P < 0.01 compared with comparative example 1.
Experimental example 4
Bamboo willow bark anti-inflammatory activity extract acute toxicity test of the present invention:
Taking 18~22g of weight Kunming mouse 20, half male and half female is randomly divided into 2 groups, and every group 20, including embodiment 1
Group (being dissolved with 10%DMSO, it is 200mg/mL test sample solution that concentration, which is made) and blank group (10%DMSO).12h before testing
Mouse is deprived of food but not water, and each group presses the dosage gastric infusion of 20mLkg-1, to the weight of mouse, behavior, four in 15d after administration
Situations such as limb activity, feed, breathing, excretion, death, is observed, and compared to the blank group, sample sets are shown no obvious abnormalities, 15d
Cervical dislocation is put to death afterwards, is observed organs such as mouse core, liver, spleen, fertilizer, kidneys, and compared to the blank group, sample sets have no obvious different
Often, show the obvious toxic-side effects for not observing bamboo willow ethyl acetate extract during observation.
Experimental example 5
The human trial of bamboo willow bark anti-inflammatory activity extract clinical application-treatment psoriasis of the present invention
1. the preparation of bamboo willow ethyl acetate extract tablet
The bamboo willow ethyl acetate extract powder 80g prepared by embodiment 1 is weighed, is crushed, 80 meshes are crossed, it is beautiful that 12g is added
Rice starch (diluent), 8g microcrystalline cellulose (adhesive and disintegrating agent) mix 45min, feed the mixture into granulator and add
Heat is granulated, and is pre-mixed 5min, when leaving air temp reaches 40 DEG C, sprays 90wt% ethanol solution, drying contains material at 50 DEG C
Water control is in 5.0wt% hereinafter, then crossing 14 mesh nylon mesh progress whole grain, the addition 0.5g stearic acid into the particle after whole grain
Magnesium (lubricant), mixes 10min, and tabletting obtains the bamboo willow ethyl acetate extract tablet that slice weight is 0.5g.
2. general information
Psoriatic 100, the age 18~65 years old, the course of disease was between 1 month to 10 years.
3. cardinal symptom
Red papules or maculopapule, syringe needle to mung bean size, clear border are above covered silvery white or the mica sample scales of skin that peel off, can be scraped
It removes, with different degrees of itch, is mainly in four autopodium sides, ancon, knee, the small of the back and scalp.
4. experimental group
Patient is randomly divided into two groups, including treatment group and control group, every group 50.Treatment group takes to make according to the above method
Standby bamboo willow ethyl acetate extract tablet is treated, 3 times a day, 1~2 every time, a month as a treatment course, control group
Outer painting Tretinoin ointment even applies a month as a treatment course 3 times a day.Conditions of patients variation is observed after 3 courses for the treatment of.
5. curative effect judging standard
Recovery from illness: symptom after taking medicine disappears；
Effective: symptom after taking medicine mitigates；
It is invalid: symptom slightly mitigation or unchanged.
Each group is shown in Table 4 to the therapeutic effect of psoriasis.Tablets have psoriasis it can be seen from experimental result
Preferable therapeutic effect (total effective rate > 90%), and its therapeutic effect is better than therapeutic effect (the total effective rate > of positive control
64%), and treatment group patient, do not occur any adverse reaction or toxicity during observation of taking medicine, show that the tablet is facing
It can be used for the treatment of psoriasis on bed.
Therapeutic effect of 4 each group of table to psoriasis
Claims (7)
1. a kind of preparation method of bamboo willow bark anti-inflammatory activity extract, characterized in that by bamboo willow bark powder, 5 to 20 times of ethyl alcohol of use
Ultrasonic extraction 1~5 time, each 30-120min obtains extracting solution；Extracting solution is concentrated under reduced pressure into no ethyl alcohol residual, distilled water is added
Dissolution, is extracted with ethyl acetate, through both obtaining after purification twice；
The preparation method of the bamboo willow bark anti-inflammatory activity extract, the specific steps are as follows:
A) extract: ethanol solution, ultrasonic extraction item is added according to solid-liquid ratio 1:5~1:20 in bamboo willow bark powder, 10~40 mesh of granularity
Part is 9~18KHz of frequency, and 20~50 DEG C of temperature, 30~120min of time, ultrasonic number 1~5 time obtains extracting solution；It extracts
Liquid is concentrated under reduced pressure into no ethyl alcohol residual, and appropriate distilled water dissolution is added, is extracted 1~3 time with 1~3 times of amount ethyl acetate, extract liquor
It is concentrated under reduced pressure, vacuum drying obtains crude extract powder；
B silica gel column chromatography: 100~300 mesh silica gel wet method dress posts, diameter height ratio 1:5, step A) is depressurized) gained powder dry method loading,
Using petroleum ether as eluant, eluent, low polar impurity is removed；Using petroleum ether and ethyl acetate volume ratio for 2:8 mixed liquor as elution
Agent, eluent are concentrated under reduced pressure into certain volume, and vacuum drying obtains the bamboo willow bark anti-inflammatory activity extract powder of initial purification；
C) Sephadex LH-20 is purified: gel column diameter height ratio 1:18, step B) the gained a small amount of chloroform-methanol mixed liquor of powder
Dissolution, wet process loading, using chloroform-methanol 1:1 as eluant, eluent, flow velocity is 0.3~0.5BVh-1, isocratic elution obtains bamboo willow bark
Activity extract solution；
D) dry and save: solution step C) is concentrated under reduced pressure into certain volume, is dried in vacuo, and after sterilized, sterilization, obtains bamboo willow
Skin activity extract powder.
2. the preparation method of bamboo willow bark anti-inflammatory activity extract described in claim 1, characterized in that the step A) in ethyl alcohol
The volume fraction of solution be 70%, raw material granularity be 24 mesh, solid-liquid ratio 1:10, supersonic frequency 18KHz, 30 DEG C of ultrasonic temperature,
Ultrasonic time 60min, ultrasonic number 3 times；The dosage of ethyl acetate is identical as the dosage of distilled water used in dissolution extract, extraction
3 times.
3. the preparation method of bamboo willow bark anti-inflammatory activity extract described in claim 1, characterized in that the step B) in silica gel
Granularity is 200~300 mesh；Using petroleum ether as eluent 2BV, mixed with volume ratio for the petroleum ether and ethyl acetate of 2:8
Liquid is eluent 3BV, merges eluent, is concentrated under reduced pressure under the conditions of 40 DEG C.
4. the preparation method of bamboo willow bark anti-inflammatory activity extract described in claim 1, characterized in that the step C) in wet process
The sample concentration of sample is 50mg/mL, flow velocity 0.4BVh-1, 3BV is eluted, the eluent after collecting 2h.
5. the preparation of any bamboo willow bark anti-inflammatory activity extract of 1-3 containing claim, characterized in that the preparation is served as reasons
Bamboo willow bark anti-inflammatory activity extract and pharmaceutically acceptable carrier or diluent composition.
6. the preparation of the anti-inflammatory activity extract of willow bark containing bamboo described in claim 5, characterized in that the preparation is tablet, sugar
Clothing ball, capsule, injection, solution, lotion, ointment, emulsifiable paste, aerosol or suppository.
7. the preparation described in claim 5 or 6 containing bamboo willow bark anti-inflammatory activity extract, in the drug of preparation prevention and treatment psoriasis
In application.
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| CN103242422A (en) * | 2013-05-15 | 2013-08-14 | 南京泽朗医药科技有限公司 | Method for extracting cyclocaric acid A from cyclocarya paliurus leaves |
| CN104688811A (en) * | 2014-12-29 | 2015-06-10 | 昆明皕凯科技有限公司 | Acne removal composition based on natural plant raw materials and application thereof |
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| CN102532220A (en) * | 2010-12-24 | 2012-07-04 | 苏州宝泽堂医药科技有限公司 | Method for separating and purifying sec-o-glucosylhamaudol |
| CN102311981A (en) * | 2011-07-19 | 2012-01-11 | 东南大学 | Method for preparing and purifying prodigiosin |
| CN103242422A (en) * | 2013-05-15 | 2013-08-14 | 南京泽朗医药科技有限公司 | Method for extracting cyclocaric acid A from cyclocarya paliurus leaves |
| CN104688811A (en) * | 2014-12-29 | 2015-06-10 | 昆明皕凯科技有限公司 | Acne removal composition based on natural plant raw materials and application thereof |
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